Collaborative interplay between FGF-2 and VEGF-C promotes lymphangiogenesis and metastasis

Interplay between various lymphangiogenic factors in promoting lymphangiogenesis and lymphatic metastasis remains poorly understood. Here we show that FGF-2 and VEGF-C, two lymphangiogenic factors, collaboratively promote angiogenesis and lymphangiogenesis in the tumor microenvironment, leading to widespread pulmonary and lymph-node metastases. Coimplantation of dual factors in the mouse cornea resulted in additive angiogenesis and lymphangiogenesis. At the molecular level, we showed that FGFR-1 expressed in lymphatic endothelial cells is a crucial receptor that mediates the FGF-2–induced lymphangiogenesis. Intriguingly, the VEGFR-3–mediated signaling was required for the lymphatic tip cell formation in both FGF-2– and VEGF-C–induced lymphangiogenesis. Consequently, a VEGFR-3–specific neutralizing antibody markedly inhibited FGF-2–induced lymphangiogenesis. Thus, the VEGFR-3–induced lymphatic endothelial cell tip cell formation is a prerequisite for FGF-2–stimulated lymphangiogenesis. In the tumor microenvironment, the reciprocal interplay between FGF-2 and VEGF-C collaboratively stimulated tumor growth, angiogenesis, intratumoral lymphangiogenesis, and metastasis. Thus, intervention and targeting of the FGF-2– and VEGF-C–induced angiogenic and lymphangiogenic synergism could be potentially important approaches for cancer therapy and prevention of metastasis.     

Adenoviral expression of vascular endothelial growth factor-C induces lymphangiogenesis in the skin

The growth of blood and lymphatic vasculature is mediated in part by secreted polypeptides of the vascular endothelial growth factor (VEGF) family. The prototype VEGF binds VEGF receptor (VEGFR)-1 and VEGFR-2 and is angiogenic, whereas VEGF-C, which binds to VEGFR-2 and VEGFR-3, is either angiogenic or lymphangiogenic in different assays. We used an adenoviral gene transfer approach to compare the effects of these growth factors in adult mice. Recombinant adenoviruses encoding human VEGF-C or VEGF were injected subcutaneously into C57Bl6 mice or into the ears of nude mice. Immunohistochemical analysis showed that VEGF-C upregulated VEGFR-2 and VEGFR-3 expression and VEGF upregulated VEGFR-2 expression at 4 days after injection. After 2 weeks, histochemical and immunohistochemical analysis, including staining for the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), the vascular endothelial marker platelet-endothelial cell adhesion molecule-1 (PECAM-1), and the proliferating cell nuclear antigen (PCNA) revealed that VEGF-C induced mainly lymphangiogenesis in contrast to VEGF, which induced only angiogenesis. These results have significant implications in the planning of gene therapy using these growth factors.     

Vascular endothelial growth factor C induces angiogenesis in vivo

Vascular endothelial growth factor C (VEGF-C) recently has been described to be a relatively specific growth factor for the lymphatic vascular system. Here we report that ectopic application of recombinant VEGF-C also has potent angiogenic effects in vivo. VEGF-C is sufficiently potent to stimulate neovascularization from limbal vessels in the mouse cornea. Similar to VEGF, the angiogenic response of corneas induced by VEGF-C is intensive, with a high density of new capillaries. However, the outgrowth of microvessels stimulated by VEGF-C was significantly longer than that induced by VEGF. In the developing embryo, VEGF-C was able to induce branch sprouts from the established blood vessels. VEGF-C also induced an elongated, spindle-like cell shape change and actin reorganization in both VEGF receptor (VEGFR)-2 and VEGFR-3-overexpressing endothelial cells, but not in VEGFR-1-expressing cells. Further, both VEGFR-2 and VEGFR-3 could mediate proliferative and chemotactic responses in endothelial cells on VEGF-C stimulation. Thus, VEGF-C may regulate physiological angiogenesis and participate in the development and progression of angiogenic diseases in addition to lymphangiogenesis.     

Involvement of vascular endothelial growth factor receptor-3 in maintenance of integrity of endothelial cell lining during tumor angiogenesis

Vascular endothelial growth factor (VEGF) plays a major role in tumor angiogenesis. VEGF-C, however, is thought to stimulate the growth of lymphatic vessels because an expression of its specific receptor, VEGF receptor-3 (VEGFR-3), was demonstrated to be restricted to lymphatic vessels. Here we demonstrate that the inactivation of VEGFR-3 by a novel blocking monoclonal antibody (mAb) suppresses tumor growth by inhibiting the neo-angiogenesis of tumor-bearing tissues. Although VEGFR-3 is not expressed in adult blood vessels, it is induced in vascular endothelial cells of the tumor-bearing tissues. Hence, VEGFR-3 is another receptor tyrosine kinase involved in tumor-induced angiogenesis. Micro-hemorrhage in the tumor-bearing tissue was the most conspicuous histologic finding specific to AFL4 mAb-treated mice. Scanning microscopy demonstrated disruptions of the endothelial lining of the postcapillary venule, probably the cause of micro-hemorrhage and the subsequent collapse of the proximal vessels. These findings suggest the involvement of VEGFR-3 in maintaining the integrity of the endothelial lining during angiogenesis. Moreover, our results suggest that the VEGF-C/VEGFR-3 pathway may serve another candidate target for cancer therapy. (Blood. 2000;96:546-553)     

环氧化酶2、血管内皮生长因子C/血管内皮生长因子受体3与肺癌淋巴转移

淋巴转移是肺癌转移途径之一.在肺癌淋巴转移过程中,血管内皮生长因子及其受体家族发挥了重要的作用.尤其是淋巴内皮特异性受体血管内皮生长因子受体3及其配体血管内皮生长因子C对促进淋巴管生长具有关键的作用.同时近年研究表明环氧化酶2与肺癌淋巴转移呈正相关.环氧化酶2、血管内皮生长因子C/血管内皮生长因子受体3有望成为肺癌淋巴管转移靶向治疗的新靶点.     

VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses

Optimal angiogenic and lymphangiogenic gene therapy requires knowledge of the best growth factors for each purpose. We studied the therapeutic potential of human vascular endothelial growth factor (VEGF) family members VEGF-A, VEGF-B, VEGF-C, and VEGF-D as well as a VEGFR-3-specific mutant (VEGF-C156S) using adenoviral gene transfer in rabbit hindlimb skeletal muscle. The significance of proteolytic processing of VEGF-D was explored using adenoviruses encoding either full-length or mature (DeltaNDeltaC) VEGF-D. Adenoviruses expressing potent VEGFR-2 ligands, VEGF-A and VEGF-DDeltaNDeltaC, induced the strongest angiogenesis and vascular permeability effects as assessed by capillary vessel and perfusion measurements, modified Miles assay, and MRI. The most significant feature of angiogenesis induced by both VEGF-A and VEGF-DDeltaNDeltaC was a remarkable enlargement of microvessels with efficient recruitment of pericytes suggesting formation of arterioles or venules. VEGF-A also moderately increased capillary density and created glomeruloid bodies, clusters of tortuous vessels, whereas VEGF-DDeltaNDeltaC-induced angiogenesis was more diffuse. Vascular smooth muscle cell proliferation occurred in regions with increased plasma protein extravasation, indicating that arteriogenesis may be promoted by VEGF-A and VEGF-DDeltaNDeltaC. Full-length VEGF-C and VEGF-D induced predominantly and the selective VEGFR-3 ligand VEGF-C156S exclusively lymphangiogenesis. Unlike angiogenesis, lymphangiogenesis was not dependent on nitric oxide. The VEGFR-1 ligand VEGF-B did not promote either angiogenesis or lymphangiogenesis. Finally, we found a positive correlation between capillary size and vascular permeability. This study compares, for the first time, angiogenesis and lymphangiogenesis induced by gene transfer of different human VEGFs, and shows that VEGF-D is the most potent member when delivered via an adenoviral vector into skeletal muscle.     

Angiogenesis and lymphangiogenesis in parathyroid proliferative lesions

Angiogenesis and lymphangiogenesis are involved in tumoral growth and metastatic spread. There is little information on angiogenesis and no available data on lymphangiogenesis in parathyroid glands (PTG). Using immunohistochemistry for CD34, LYVE-1 (specific markers for vascular and lymphatic endothelium, respectively), vascular endothelial growth factor (VEGF)-A, VEGF-C, and fibroblast growth factor (FGF)-2, this study analyzes microvascular density (MVD), lymphatic vascular density (LVD), and expression of angiogenic and lymphangiogenic growth factors in 13 normal PTG, 77 parathyroid adenomas (PTA), and 17 primary parathyroid hyperplasia (PPH). MVD was higher in PPH and PTA, compared with PTG (P < 0.001). There was no difference in VEGF-A expression among groups. In contrast, FGF-2 expression was higher in PPH, compared with PTA and PTG (P < 0.0001). FGF-2 scores and MVD were significantly correlated (r = 0.43). LVD did not differ among groups, and VEGF-C expression was unrelated to LVD. There was no relationship between MVD and tumor behavior (adenoma size, PTH, or calcium). In conclusion, this study shows increased angiogenesis in parathyroid proliferative lesions compared with normal glands and suggests that FGF-2 is proangiogenic in parathyroid tissue. In PTA, tumor behavior is not related to angiogenic phenotype. This is the first demonstration of lymphatic vessels in PTG, but the lack of correlation with VEGF-C expression suggests that VEGF-C is not the primary lymphangiogenic factor.     

The biology of vascular endothelial growth factors

The discovery of the vascular endothelial growth factor (VEGF) family members VEGF, VEGF-B, placental growth factor (PlGF), VEGF-C and VEGF-D and their receptors VEGFR-1, -2 and -3 has provided tools for studying the vascular system in development as well as in diseases ranging from ischemic heart disease to cancer. VEGF has been established as the prime angiogenic molecule during development, adult physiology and pathology. PlGF may primarily mediate arteriogenesis, the formation of collateral arteries from preexisting arterioles, with potential future therapeutic use in for example occlusive atherosclerotic disease. VEGF-C and VEGF-D are primarily lymphangiogenic factors, but they can also induce angiogenesis in some conditions. While many studies have addressed the role of angiogenesis and the blood vasculature in human physiology, the lymphatic vascular system has until recently attracted very little attention. In this review, we will discuss recent advances in angiogenesis research and provide an overview of the molecular players involved in lymphangiogenesis.     

Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1–3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4–7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs.     

肿瘤抗淋巴管生成研究进展

肿瘤转移是癌症患者的主要死因之一,淋巴管转移是肿瘤转移的重要途径,研究发现VEGF-C/VEGF-D/VEGFR-3与肿瘤淋巴管生成、肿瘤转移、肿瘤预后密切相关.大量的研究证实VEGF-C/VEGF-D/VEGFR-3信号传导轴在调节肿瘤淋巴管生成中起主导作用,临床病理研究也显示VEGF-C/VEGF-D/VEGFR-...     

Vascular endothelial growth factor-C: a growth factor for lymphatic and blood vascular endothelial cells

The endothelial cells lining all vessels of the circulatory system have been recognized as key players in a variety of physiological and pathological settings. They act as regulators of vascular tone via the inducible nitric oxide system and in angiogenesis, the formation of blood vessels de novo. Aberrant regulation of endothelial cells contributes to tumor formation, atherosclerosis, and diseases such as psoriasis and rheumatoid arthritis. Among the most recently discovered growth factors for endothelial cells are newly isolated members of the platelet-derived growth factor/vascular endothelial growth factor (VEGF) family, VEGF-B, VEGF-C, and VEGF-D. VEGF-C is the ligand for the receptor tyrosine kinase VEGFR-3 (also known as Flt4), which is expressed predominantly in lymphatic endothelium of adult tissues, but a proteolytically processed form of VEGF-C can also activate VEGFR-2 of blood vessels. The lymphatic vessels have been known since the 17th century, but their specific roles in health and disease are still poorly understood. With the discovery of VEGF-C and its cognate receptor VEGFR-3, the regulation and functions of this important component of the circulatory system can be investigated.     

Expression of vascular endothelial growth factor-C and the relationship between lymphangiogenesis and lymphatic metastasis in colorectal cancer

AIM: To investigate the expression of vascular endothelial growth factor-C (VEGF-C) and the relationship between VEGF-C and lymphangiogenesis, lymph node metastasis in colorectal cancer. METHODS: Fifty six cases of colorectal cancer were selected randomly. Expression of VEGF-C was detected by immunohistochemistry, and lymphatic vessels were stained by enzyme histochemical method. RESULTS: VEGF-C expression was found in 66.7% (37/56) patients. In VEGF-C positive and negative patients, the lymphatic vessel density was 25.16+/-7.52 and 17.14+/-7.22, respectively (P<0.05). The rate of lymph node metastasis in VEGF-C positive patients (81.1%) was significantly higher than that in the negative group (42.1%). CONCLUSION: VEGF-C expression may induce lymphangiogenesis in colorectal cancer, as a result, tumor cells can entry the lymphatic vessels easily. VEGF-C may serve as a useful prognotic factor in colorectal carcinoma.     

血管内皮生长因子C及受体与胃癌淋巴转移关系的研究

目的探讨血管内皮生长因子-C(VEGF-C)及其受体-3(VEGFR-3)在胃癌中的表达及其与淋巴结转移的关系。方法检测延安大学附属医院2012年1月-2013年1月收治的78例原发性胃癌患者胃黏膜VEGF-C和VEGFR-3蛋白的表达,并与20例胃癌患者经病理证实为正常胃黏膜的远切端组织进行比较,分析胃癌淋巴结转移情况。结果 VEGF-C在正常胃黏膜组织中呈阴性表达,在胃癌组织中定位于癌细胞胞浆中,呈弥散性分布,78例胃癌组织中,60例表达阳性,阳性率为76.92%,胃癌组织与正常胃黏膜组织VEGF-C阳性率差异有统计学意义(P0.01);VEGF-C的表达与组织学分级、浸润深度、有无淋巴转移及TNM分期密切相关。VEGFR-3在正常胃黏膜细胞中着色均匀,主要分布于细胞胞浆内,阳性表达率为10.00%;在胃癌组织中的表达异质明显,呈灶状或弥散性分布,阳性率为66.67%,差异有统计学意义(P0.01);VEGFR-3表达与组织学分级、有无淋巴转移及TNM分期密切相关。VEGF-C与VEGFR-3表达呈正相关。结论 VEGF-C及其受体VEGFR-3在胃癌的发生、发展及淋巴转移中发挥着重要作用。     

COX-2 expression is correlated with VEGF-C, lymphangiogenesis and lymph node metastasis in human cervical cancer

Lymphangiogenesis has been shown to promote lymph node metastasis in cancers, making it an important target in cancer therapy. Vascular endothelial growth factor (VEGF)-C is upregulated in various tumors/cancers and is one of the most potent growth factors for inducing lymphangiogenesis and promoting lymph node metastasis (LNM). Likewise, cyclooxygenase (COX)-2 plays major roles in carcinogenesis, tumor growth and metastasis via multiple mechanisms including inactivation of host antitumor immunity and promotion of tumor cell migration, tumor cell invasiveness and tumor-associated angiogenesis and lymphangiogenesis. We previously demonstrated an association between COX-2 and VEGF-C in an in vitro model of lung cancer. However, little is known about the regulation of VEGF-C by COX-2 in cervical cancer. In this study, we measured the COX-2 and VEGF-C expressions by immunohistochemistry in 23 LNM-positive and 20 LNM-negative cervical cancer specimens. We then examined the correlations among the expressions and the lymphatic microvessel density (LMVD) and ultrastructural changes to the lymphatic vessel walls by enzyme histochemical staining and electron microscopy. In addition, we used the HeLa cervical cancer cell line to explore the in vitro regulation of VEGF-C by COX-2 and its metabolite, PGE₂, using siRNA-mediated gene silencing and EP receptor blockade. The LNM-positive specimens exhibited significantly higher VEGF-C expression, COX-2 expression and LMVD than the LNM-negative specimens. Furthermore, there were strong correlations between the levels of COX-2 expression and the levels of VEGF-C expression and secretion and a significant positive association between the LMVD and LNM. siRNA-mediated knockdown of COX-2 expression inhibited VEGF-C mRNA expression while EP1 and EP4 receptor antagonists reduced the VEGF-C protein level and tyrosine phosphorylation of Src kinase. Moreover, inhibition of Src kinase with the tyrosine kinase inhibitor PP1 attenuated VEGF-C expression. Collectively, our data provide evidence for a clinical association between COX-2 and VEGF-C expressions in cervical cancer. EP1 and EP4 receptors may be involved in the COX-2-mediated regulation of VEGF-C protein and mRNA expressions. Src may be a downstream mediator of EP1 and EP4 receptors. COX-2 inhibition may diminish LNM by suppressing VEGF-C-mediated lymphangiogenesis.     

Comparative evaluation of FGF-2-, VEGF-A-, and VEGF-C-induced angiogenesis, lymphangiogenesis, vascular fenestrations, and permeability

Several endothelial growth factors induce both blood and lymphatic angiogenesis. However, a systematic comparative study of the impact of these factors on vascular morphology and function has been lacking. In this study, we report a quantitative analysis of the structure and macromolecular permeability of FGF-2-, VEGF-A-, and VEGF-C-induced blood and lymphatic vessels. Our results show that VEGF-A stimulated formation of disorganized, nascent vasculatures as a result of fusion of blood capillaries into premature plexuses with only a few lymphatic vessels. Ultrastructural analysis revealed that VEGF-A-induced blood vessels contained high numbers of endothelial fenestrations that mediated high permeability to ferritin, whereas the FGF-2-induced blood vessels lacked vascular fenestrations and showed only little leakage of ferritin. VEGF-C induced approximately equal amounts of blood and lymphatic capillaries with endothelial fenestrations present only on blood capillaries, mediating a medium level of ferritin leakage into the perivascular space. No endothelial fenestrations were found in FGF-2-, VEGF-A-, or VEGF-C-induced lymphatic vessels. These findings highlight the structural and functional differences between blood and lymphatic vessels induced by FGF-2, VEGF-A, and VEGF-C. Such information is important to consider in development of novel therapeutic strategies using these angiogenic factors.     

淋巴管生成的分子机制与恶性肿瘤的转移

长期以来一直认为淋巴管是恶性肿瘤转移的有效途径;但是,关于淋巴管内皮细胞的特异性标记物缺乏一致的资料,而且,恶性肿瘤内是否存在新生淋巴管也一直有争议。直到最近,发现了新的淋巴管内皮细胞标记物,在肿瘤的动物模型以及人类肿瘤中均发现了新生淋巴管,对于VEGF-C、D／VEGFR-3信号途径的深入了解,因此淋巴管生成在肿瘤转移中的相关研究得以深入进行。越来越多的研究资料表明,淋巴管生成同肿瘤的转移播散、肿瘤患者的预后等直接相关。现将淋巴管生成的分子机制以及其与肿瘤转移的相关研究进展综述。     

Lymphangiogenesis in aortic valve stenosis--novel regulatory roles for valvular myofibroblasts and mast cells

ObjectiveTo investigate mechanisms of lymphangiogenesis in aortic valve stenosis (AS).MethodsLymphatic vessels were visualized with LYVE-1 staining in 20 control, 5 sclerotic, and 40 stenotic human aortic valves. Vascular endothelial growth factors (VEGFs) VEGF-C and VEGF-D, and their lymphangiogenic receptor VEGFR-3, and the angiogenic VEGFR-2 were analysed by quantitative real-time PCR and immunohistochemistry. Cultured myofibroblasts derived from human stenotic aortic valves, and cultured human mast cells were used to study VEGF-C regulation, and VEGF-C and VEGF-A were quantified from cell culture media by enzyme immunoassays.ResultsLymphatic vessels, VEGF-C, VEGF-D, VEGFR-3 and VEGFR-2 all were present in the aortic valves. In AS, the number of lymphatic vessels and the expression of VEGF-D, VEGFR-3, and VEGFR-2 were increased. Moreover, the numbers of lymphatic vessels correlated positively with those of neovessels (r = 0.525, p = 0.001) and mast cells (r = 0.374, p = 0.017). Cultured valvular myofibroblasts produced VEGF-C, and addition of tumour necrosis factor alpha (TNF-α) to the cells augmented its secretion. In contrast, proteases released by activated human mast cells degraded VEGF-C.ConclusionThese results show that lymphangiogenesis is induced in advancing AS. Furthermore, valvular myofibroblasts and activated mast cells were identified as novel regulators of lymphangiogenesis in aortic valves.